Effects of curcumin on the growth performance, apparent nutrient digestibility, intestinal morphology, digestive enzyme activity, and antioxidant capacity of meat rabbits

Abstract The purpose of this study was to investigate the effect of curcumin on the growth performance, slaughter performance, apparent nutrient digestibility, intestinal morphology, and digestive enzyme activity of meat rabbits. One hundred and sixty healthy weaned meat rabbits were randomly divided into four groups (forty replicates per group and one rabbit per replicate). The control group was fed a basal diet and the test groups (T1, T2 and T3) were fed diets supplemented with 50, 100 and 150 g/t curcumin in the basal diet, respectively. The study lasted for 28 days, including a pre-feeding period of 7 days and a formal trial period of 21 days. The results showed that diets supplemented with curcumin significantly increased rabbits’ body weight gain and average daily feed intake; Apparent digestibility of energy, crude protein, crude fat, crude fibre, NDF and ADF; Jejunal villus height, the ratio of villus height to crypt depth; The activities of amylase and lipase in jejunal and caecal digesta and cellulase in caecal digesta; Caecal mucosal thickness, eviscerated dressing percentage, and dorsal and abdominal hair thickness (p < .05). Diets supplemented with curcumin significantly decreased rabbits’ feed to gain ratio, jejunal crypt depth and thickness of the intestinal wall (p < .05). In conclusion, curcumin is a kind of valuable feed additive, which can improve the nutrient digestibility, growth performance, slaughter ratio and fur development of meat rabbits by improving digestive tract development and digestive enzyme activity. The appropriate dosage in the meat rabbit diet is 100 g/t. HIGHLIGHTS Dietary supplementation of curcumin improved the growth performance, gastrointestinal digestive enzyme activities and slaughter performance of meat rabbits. Dietary supplementation of curcumin increased the antioxidant capacity of meat rabbits. Curcumin could be used as a new kind of feed additive for meat rabbits.


Introduction
Rabbit is delicious and appreciated by many people around the world.During the weaning period, the digestive tracts of meat rabbits are not fully developed and are subjected to multiple stresses at the same time, such as weaning, changing food and even cage transfers, which may affect the growth and development of meat rabbits (Bivolarski and Vachkova 2014).
Plant extracts, as a kind of feed additive with specific physiological functions, have been paid more and more attention in recent years (Kon e et al. 2019).Studies have found that the active plant extracts mainly include plant flavonoids, saponins, polysaccharides, etc (Shehata et al. 2022).A large number of experiments have confirmed that they can play a significant positive regulatory role in regulating the balance of intestinal microorganisms, improving animal immunity, promoting intestinal development, and improving the ability to anti-stress animals (Attia et al. 2020;Corbi et al. 2018;Hassan et al. 2020;Imbabi et al. 2021).
Curcumin from Curcuma longa was first isolated in small molecules poly-phenols compounds.Curcumin has anti-inflammatory, antioxidant, lipid, antivirus, antiinfection, anti-tumor, anticoagulation, and liver fibrosis, such as a wide range of pharmacological activities of atherosclerosis, and low toxicity, low adverse reactions (Kotha and Luthria 2019).It is one of the world's biggest natural edible pigments and a food additive approved by the World Health Organisation, the Food and Drug Administration of the United States and many countries.Curcumin, as a non-steroidal anti-inflammatory drug, has a wide range of preventive properties against diseases (Patel et al. 2020).Since modern medical research has found that the occurrence of many diseases in the human body is related to the formation of free radicals and the participation of inflammatory reactions, curcumin antioxidant activity and anti-inflammatory effect have attracted extensive attention (Kahkhaie et al. 2019;Llano et al. 2019).In recent years, there have been a number of research reports on the application of curcumin to pigs, chickens and aquaculture with positive results, but there are fewer reports on curcumin as a feed additive in meat rabbit production, which limits its application scale in meat rabbit production (Attia et al. 2017).Therefore, this study of curcumin investigates the effects of curcumin on growth performance, gastrointestinal digestive enzyme activity, serum biochemical parameters, immunity, slaughter performance and meat quality of meat rabbits, which provides a scientific reference for the rational application of curcumin in rabbit production.

Animals and experimental design
In the present study, the curcumin (C 21 H 20 O 6 , a bis-a, b-unsaturated b-diketone) (>95% purity) was provided by Fudi Biotechnology Company Limited (Beijing, China).In this study, one hundred and sixty 35-days healthy weaned lra rabbits with suitable weight (1130 ± 18 g) were randomly divided into four groups (each group had 20 males and 20 females, 40 replicates per group and one rabbit per replicate, each group was randomly distributed according to the average weight).The basal diet was free of antibiotics and mildew inhibitors.The control group was fed a basal diet and the experimental groups (T1, T2 and T3) were fed diets supplemented with 50 g/t,100g/t and 150 g/t curcumin in the basal diet, respectively.The T1 means 50 g/t curcumin addition group, T2 means 100 g/t curcumin addition group, and T3 means 150 g/t curcumin addition group.The rabbits were fed the diets according to the standards of National Research Council (NRC) (2012).The ingredients and chemical composition of the diets are shown in Table 1.
The feeding experiment lasted for 28 days, including a pre-feeding period of 7 days and a formal trial period of 21 days.Before the feeding experiment, the experiment equipment's (including cages, water nipples and feeders etc) was disinfected.During the feeding experiment, the meat rabbits ate and drank freely and fed twice a day, and each rabbit lived in a single cage.The natural light and ventilation were maintained as well as the rabbits were immunised according to rabbit vaccination regulations.

Growth indices
During the feeding experiment, meat rabbits were weighed in the morning before a meal on day 1 and day 21, respectively.The feed intake was counted on the first and last day, and the average daily gain (ADG), average daily feed intake (ADFI) and feed-toweight ratio (feed/gain) was calculated.
Slaughter performance and organ index At the end of the feeding experiment, after the meat rabbits fasted for 12 h, eight rabbits (four male rabbits and four female rabbits) were randomly selected from each group.After each rabbit was euthanized, the rabbit was weighed before slaughter.The carcase weight and full eviscerated slaughter weight were recorded after each rabbit was slaughtered to calculate the slaughter ratio, full eviscerated slaughter ratio and half eviscerated slaughter ratio.
The carcase weight refers to the weight of the rabbit after the blood, limbs, fur, tail, gastrointestinal and genitourinary tract were removed.The full eviscerated slaughter weight was the carcase weight with the head and all internal organs removed.
The organ index was calculated as the ratio of organ weight over the entire body weight: organ index (g/kg) ¼ organ weight (g)/body weight (kg).

Meat quality and wool thickness
The leg and back muscles were taken from both right sides after each rabbit was slaughtered and the pH was measured at 45 min and 24 h after slaughtering with a Testo 205 pH metre (Testo Company, Lenzkirch, Germany).
Using vernier calliper, the thickness of 1 cm width coat was measured in the shoulder, back, abdomen and buttocks of meat rabbits.

Apparent Nutrient digestibility
After the growth test, 8 rabbits (male and female half) were randomly selected from each group, and transferred to the digestive and metabolic cage for a digestion test (7 days).The experiment adopted the method of total faeces collection.During the period, the feed intake of experimental rabbits was recorded every day, and all faeces were collected.After weighing, they were divided into two parts.One part was added with 10% hydrochloric acid for the nitrogen fixation to determine the crude protein, and the other part was used to determine other nutrient contents.
After the experiment, the dung samples were dried and crushed and put in a sample bag for testing.The nutrient content in the diet and dung were determined.
Apparent digestibility (%) of a certain nutrient ¼ (the nutrient content in diet-the nutrient content in faeces)/the nutrient content in diet Â 100.

Intestinal morphology
On the 21st day of the trial period, eight experimental rabbits were selected from each group, and slaughtered, and every experimental rabbit took 1 cm jejunum and caecum segment for the fixed test.The images of intestinal sections were collected by BA210 digital camera system, and the villi height, crypt depth and wall thickness of jejunum were measured.Each section was measured 10 times, the average value was taken, and the ratio of villi height to recess depth was calculated.

Gastrointestinal digestive enzyme activity
On the 21st day of the trial period, eight rabbits in each group were selected and slaughtered.After slaughter, 5 mL chyme in jejunum and caecum were put into a freezing tube and stored at À80 C. The activities of a-amylase and lipase of chyme in jejunum and caecum were detected using the kit (produced by Nanjing Jiancheng Biotechnology Research Institute, Nanjing, China), which was strictly operated in accordance with the kit instructions.The cellulase activity of caecum chyme was detected by 3,5-dinitrosalicylic acid (DNS) method.The pH of caecum chyme was measured with pH-3b acidimeter.

Statistical analysis
Excel 2019 and SPSS software package (version 20) was used for data statistical analysis (for growth performance analysis, n ¼ 40; for gastrointestinal digestive enzyme activity, apparent nutrient digestibility, intestinal structure, slaughter performance, meat quality and wool thickness analysis, n ¼ 8).All the data were analysed by a one-way ANOVA model, followed by Duncan's multiple range tests.When a significant difference was observed, the difference among the groups was assessed (p < .05).Statistical modal: Yijk ¼ l þ Ti þ eij, where Yijk ¼ an observation, l ¼ the overall mean, Ti ¼ effect of treatments, and eij ¼ random error.

Apparent nutrient digestibility
As shown in

Gastrointestinal digestive enzyme activity
As shown in The pH of caecal chyme was decreased by 3.70% (p>.05), 2.74% (p>.05) and 6.16% (p>.05), respectively.It indicated that curcumin could improve the activities of digestive enzymes for the digestion and absorption of nutrients.

Organ indexes
The effect of curcumin on the organ indexes of meat rabbits is shown in Table 5.There were no significant differences in cardiac, liver, spleen, renal, thymus and sacculus rotundus index among all groups (p>.05).

Meat quality and coat density
Table 5 shows that curcumin has no significant effect on the pH of leg and back muscles at 45 min and 24 h.Compared with the control group, the wool thicknesses in the shoulder of T1, T2 and T3 groups were increased by 15.05% (p < .05),13.97% (p < .05)and 16.13% (p < .05),respectively (p>.05).The wool thicknesses in back of T1, T2 and T3 groups was increased by 3.64% (p>.05), 1.82% (p < .05)and 5.45% (p>.05), respectively.The wool thickness in the abdomen of T1, T2 and T3 groups was increased by 6.25% (p>.05), 18.75% (p < .05)and 14.58% (p < .05),respectively.The results indicated that curcumin could promote wool follicle development, increase coat density and improve wool quality to a certain extent.

Discussion
The results implied that the dietary supplementation with curcumin the growth performance of meat rabbits, which is because curcumin can improve the structure and morphology of jejunum and caecum of meat rabbits, improve the activity of digestive enzymes, and promote the digestion and absorption of nutrients.Some experiments confirmed that curcumin had a positive effect on amending intestinal integrity, promoting intestinal villi development and digestive enzyme activities.Curcumin regulates the intestinal  The organ index was calculated as the ratio of organ weight over the entire body weight: organ index (g/kg) ¼ organ weight (g)/body weight (kg).barrier function mainly by regulating intestinal epithelial permeability and intestinal flora.Curcumin protects the intestinal mucosal epithelial cells by regulating the IEL subgroup in the intestine.In addition, curcumin could promote mitochondrial autophagy and improve mitochondrial function through AMPL-TFEB signalling pathway to fight against intestinal epithelial damage (Cao et al. 2020).It was reported that curcumin could increase the content of ZO-1 and Claudin-1 in colonic epithelial cells, reduce the permeability of cell bypass, and improve intestinal barrier function (Ghosh et al. 2014).It has been verified that curcumin could reduce the bacterial endotoxin LPS and IL-1b, and further reduce the degradation of ZO-1, Claudin-1, Claudin-7 and the actin filament, thereby maintaining the integrity of the intestinal barrier (Wang et al. 2017).Tian et al. (2016) also demonstrated that curcumin could repair the intestinal epithelial structure and ensure intestinal integrity by promoting the expression of ZO-1 protein.
Some studies confirmed that curcumin could improve the health status of the intestinal structure, increase digestibility and promote growth performance.Xun et al. (2015) reported that the addition of curcumin could improve the nutrient digestibility for piglets, as well as the blood parameters and intestinal health status of piglets.The 400 mg/kg addition of curcumin could enhance the growth performance, maintain the integrity of the jejunal mucosal barrier and stimulate the immune system of the weaned piglets (Moniruzzaman et al. 2021).Curcumin supplementation could promote the growth of weaned piglets, and improve feed efficiency, immunity and antioxidant capacity (Shi et al. 2020).The aquaculture experiments of carp and tilapia showed that the addition of curcumin could promote growth and improve feed utilisation, improve the antioxidant status and enhance their immune ability (Attia et al. 2017;Moniruzzaman and Min 2020).
The present study indicated that the addition of curcumin made the jejunum villi of meat rabbits grow longer, and the crypts become shallow.The villi height to crypt depth ratios increment indicated that the intestinal absorption area becomes large, and the intestinal structural integrity becomes more complete.In addition, we found that the experimental group of meat rabbits had a thinner intestinal wall and higher jejunum and ileum digestive enzyme activities, which indicated that microflora adhered to the intestinal wall fewer and intestinal digestion and absorption capacity improved.The intestinal tract secretes digestive enzymes to digest and absorb the most important parts of feed nutrients (Hoyle 1997), and the intestinal structure, intestinal villus development and digestive enzyme activity are the main factors affecting the digestion of feed nutrients (Zhang et al. 2019).These changes increased the apparent digestibility of dietary energy and major nutrients (crude protein, ether extract and fibre), which improved the growth performance and feed conversion rate of meat rabbits.This is consistent with other test results.
The safety of feed and feed additives is the premise to ensure the safety of livestock products and human health.It is generally believed that curcumin has been eaten by humans for a long time and has low toxicity.Turmeric is currently listed as "recognized safety" (GRASS) by the United States Food and Drug Administration (FDA) as a colouring and flavouring agent in food (Sharifi-Rad et al. 2020).The safety of curcumin has been evaluated in certain studies.A study conducted by the University of Leicester in the UK showed that patients with advanced colorectal cancer had no adverse reaction after oral administration of curcumin (180 mg/d) for four months (Wahlstr€ om and Blennow 1978).Sharma's report confirmed that with the addition of 2% curcumin to the diet of rats, only minimal (nanomoles) curcumin was detected in plasma, liver and colonic mucosal tissues (Sharma et al. 2007).The safety evaluation of dimethyl curcumin (DC) by Krishnaraju et al. (2009) confirmed that the acute oral median lethal dose (LD50) of DC for female SD rats was over 5000 mg/kg, and the acute dermal LD50 was over 2000 mg/kg.No body weight change or adverse effect was observed at the autopsy.Dandekar et al. (2010) studied curcumin and found no adverse effect after long-term administration at twice the therapeutic dose.The present study confirmed that the addition of curcumin had no adverse effect on the heart, liver, spleen, kidney, thymus, and sacculus rotundus development of meat rabbits, indicating that curcumin was safe for feeding in meat rabbits.
Slaughter performance is highly correlated with meat production and directly affects the economic benefits.Slaughter rate and full eviscerated slaughter ratio are the main indicators to measure the meat production performance of animals.Oke (2018).showed that curcumin could significantly increase the final weight and improve the slaughtering performance of broilers.A study by Janz et al. (2007) showed that the addition of 500 mg/kg of ginger oil had no effect on the slaughter ratio of pigs.Curcumin supplementation had no significant effect on the slaughter performance of broilers (Abd El-Hack et al. 2021).The present study showed that curcumin could improve the slaughter ratio and the full eviscerated slaughter ratio of meat rabbits but there was no significant difference, which was similar to the other experimental results.
The wool development can also be used as an indicator reflecting the health status of meat rabbits.Within some certain, the well-developed coat indicates that the meat rabbit was in good health condition.The present study showed that the wool thickness on the shoulder, back and abdomen of meat rabbits in the experimental group was higher than that in the control group, indicating that curcumin could improve the health status of meat rabbits.
Meat quality affects the consumption and potential value of meat, and pH value is one of the important physicochemical indexes for evaluating meat quality, which is related to the shelf life, tenderness, colour and water-holding capacity of meat (Grashorn 2010).Curcumin had no significant effect on pH of broiler chicks (Pornanek and Phoemchalard 2020).The present study results were similar to those of others.

Conclusion
The present study showed that curcumin can improve growth performance, and intestinal structure and increase the activity of some digestive enzymes, it had no adverse effect on the meat quality of meat rabbits.It can be seen that curcumin could be used as a new feed additive for meat rabbits.Under this experimental condition, 100 g/t was considered optimal.
a,b Means in a row without the same superscript are different significantly (p < .05).Values are means ± standard deviation.

Table 1 .
Composition and nutrient levels of the basal diet (air-dry basis).

Table 2 .
Effect of curcumin on the growth performance of meat rabbits (n ¼ 40).

Table 3 .
Effect of curcumin on energy and nutrient apparent digestibility, digestive enzymes and pH of intestinal chyme in meat rabbits (n ¼ 8).

Table 4 .
Effect of curcumin on jejunum and caecum morphology of meat rabbits (n ¼ 8).

Table 5 .
Effect of curcumin on organ indexes, slaughter performance, meat quality and coat density of meat rabbits (n ¼ 8).